Water Research 223 (2022) 118970

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Water Research
journal homepage: www.elsevier.com/locate/watres

Bacterial and viral fecal indicator predictive modeling at three Great Lakes
recreational beach sites
Mike Cyterski a, *, Orin C. Shanks b, Pauline Wanjugi c, Brian McMinn b, Asja Korajkic b,
Kevin Oshima b, Rich Haugland b
a
U.S. Environmental Protection Agency, Office of Research and Development, Athens, GA, 30605, United States
b
U.S. Environmental Protection Agency, Office of Research and Development, Cincinnati, OH 45268, United States
c
New York State Department of Health, Center for Environmental Health, Bureau of Water Supply Protection, New York City Watershed Section, Albany, NY 12201,
United States

A R T I C L E I N F O A B S T R A C T

Keywords: Coliphage are viruses that infect Escherichia coli (E. coli) and may indicate the presence of enteric viral pathogens
Great Lakes in recreational waters. There is an increasing interest in using these viruses for water quality monitoring and
Coliphage forecasting; however, the ability to use statistical models to predict the concentrations of coliphage, as often done
qPCR
for cultured fecal indicator bacteria (FIB) such as enterococci and E. coli, has not been widely assessed. The same
Fecal indicators
Statistical modeling
can be said for FIB genetic markers measured using quantitative polymerase chain reaction (qPCR) methods.
Here we institute least-angle regression (LARS) modeling of previously published concentrations of cultured FIB
(E. coli, enterococci) and coliphage (F+, somatic), along with newly reported genetic concentrations measured
via qPCR for E. coli, enterococci, and general Bacteroidales. We develop site-specific models from measures taken
at three beach sites on the Great Lakes (Grant Park, South Milwaukee, WI; Edgewater Beach, Cleveland, OH;
Washington Park, Michigan City, IN) to investigate the efficacy of a statistical predictive modeling approach.
Microbial indicator concentrations were measured in composite water samples collected five days per week over
a beach season (~15 weeks). Model predictive performance (cross-validated standardized root mean squared
error of prediction [SRMSEP] and R2PRED) were examined for seven microbial indicators (using log10 concen­
trations) and water/beach parameters collected concurrently with water samples. Highest predictive perfor­
mance was seen for qPCR-based enterococci and Bacteroidales models, with F+ coliphage consistently yielding
poor performing models. Influential covariates varied by microbial indicator and site. Antecedent rainfall, bird
abundance, wave height, and wind speed/direction were most influential across all models. Findings suggest that
some fecal indicators may be more suitable for water quality forecasting than others at Great Lakes beaches.

1. Introduction
Statistical models have seen increased use for predicting water
quality in recreational waters in recent years (Christensen et al., 2021;
Francy et al., 2020; Jones et al., 2013). This trend is partly due to the
realization that persistence models (using water quality measures taken
one day to predict quality on the following day) are often inaccurate
(Whitman and Nevers, 2008), and waiting 8–24 h for cultured micro­
organisms to be counted (USEPA, 2021) reduces the level of public
health protection in recreational waters (Wymer et al., 2021). Statistical
predictive models could allow for public advisories to be issued quickly
enough to inform beach attendance on the same day of use (Brooks et al.,
2013).
Recreational water quality fecal indicator data, such as cultured
enterococci and E. coli, are often modeled using least squares fitting via a
multiple linear regression framework (de Brauwere et al., 2014).
Least-angle regression with Lasso (LARS-lasso, Efron et al. (2004))
modifies the linear regression approach by constraining the sum of ab­
solute regression coefficients (i.e., L1 regularization), providing a
method for identifying important/unnecessary covariates, filtering
highly collinear covariates, and reducing the overfitting of training data.
Linear regression-based methods, although sometimes outperformed by
machine learning techniques like random forests and boosted models
(Brooks et al., 2016; Thoe et al., 2012), may be less susceptible to

* Corresponding author.
E-mail address: cyterski.mike@epa.gov (M. Cyterski).

https://doi.org/10.1016/j.watres.2022.118970
Received 2 March 2022; Received in revised form 8 August 2022; Accepted 9 August 2022
Available online 10 August 2022
0043-1354/Published by Elsevier Ltd. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).
M. Cyterski et al.
overfitting with smaller datasets (n < 100) that are typically available
for recreational water quality forecast modeling.
Although cultured fecal indicator bacteria (FIB) predictive modeling
is well established, research suggests that viruses are a more likely
causative agent of many recreational waterborne illnesses compared to
bacterial pathogens (Begier et al., 2008; Sinclair et al., 2009; Soller et al.,
2010). As a result, scientists are investigating the use of coliphage as an
alternative fecal indicator for water quality testing. Coliphage (F+ and
somatic) are viruses that infect coliform bacteria, including E. coli, and
may be effective indicators of the presence of human fecal contamina­
tion and the associated risk from enteric viruses because they are
consistently found in municipal sewage and are similar in size and
structure to some human enteric viruses (Havelaar et al., 1993; King
et al., 2011; McMinn et al., 2017a). Coliphage are found in the digestive
systems of humans and other warm-blooded animals (McMinn et al.
2017a) and are routinely identified in sewage (Ewert and Paynter, 1980;
Gantzer et al., 1998; Korajkic et al., 2020; Lucena et al., 2004). Coli­
phage are also accepted metrics for microbial monitoring of ground­
water sources of drinking water (USEPA, 2006).
In addition, there is a growing interest in using genetic methods for
recreational beach monitoring. The primary advantage of these methods
over culture-based protocols is the ability to provide water quality in­
formation within a few hours (Griffith and Weisberg, 2011). Several
epidemiological studies report a significant relationship between the
incidence of swimming-related illnesses and genetic enterococci con­
centration estimates determined by quantitative polymerase chain re­
action (qPCR) methods in both marine and Great Lakes recreational
waters (Colford et al., 2012; Wade et al., 2008; Wade et al., 2010). Based
on these findings, the United States Environmental Protection Agency
(EPA) has suggested Beach Action Values associated with qPCR-based
EPA Method 1611 for enterococci (USEPA, 2012a, 2012b). A health
relationship has also been demonstrated for qPCR estimates of total
Bacteroidales by EPA Method B (USEPA, 2010c) in marine waters (Wade
et al., 2010).
This study implements LARS-lasso models and uses cross-validation
to compare the predictive performance of previously-reported (Wan­
jugi et al., 2018) coliphage (F+ and somatic), cultured FIB (E. coli and
enterococci), and newly-presented (this study) qPCR-based genetic
marker (E. coli, enterococci and Bacteroidales) concentrations for three
Great Lakes sites across a single recreational beach season (15-weeks).
Covariates include previously reported (Wanjugi et al., 2018) paired
measurements of common water and beach area parameters routinely
used to describe and model water quality conditions in recreational
settings ranging from water temperature to rainfall (Francy, 2009;
Francy et al., 2013; USEPA, 2010a, 2010b). Emphasis is placed on the
comparative predictive performance of each fecal indicator response
Fig. 1. Three Great Lakes beaches sampled for microbial water quality: Edgewater Beach in Cleveland, OH (A), Grant Park in South Milwaukee, WI (B), and
Washington Park in Michigan City, IN (C). Modified from Wanjugi et al. (2018).
2
Water Research 223 (2022) 118970
variable utilizing a standardized modeling approach. In addition, the
most influential covariates are identified to reveal potential trends that
could inform future recreational water quality sample testing and pre­
dictive modeling efforts. Findings suggest that some fecal indicators may
be more suitable for water quality forecasting than others at Great Lakes
beaches.
2. Materials and methods
2.1. Site descriptions
Sites included Edgewater Beach near Cleveland, OH, Grant Park in
South Milwaukee, WI, and Washington Park in Michigan City, IN (Fig. 1,
modified from Wanjugi et al., 2018). All are routinely monitored and
have yielded FIB concentrations that exceed the USEPA’s recommended
Beach Action Values in 10–30% (USEPA, 2012b) of samples based on
historical monitoring. At these sites, potential fecal pollution sources are
a mixture of nearby wastewater treatment facilities, stormwater runoff,
tributary inflows, and combined sewer overflows, with secondary in­
fluence from beachgoers, wildlife, and agricultural runoff. For addi­
tional details, see Wanjugi et al. (2018).
2.2. Water sampling
Water samples for microbial indicator testing were collected from
late May to early September of 2015. Each sampling event produced a 6
L composite created from six 1 L grab samples collected in a transect
area (three shin-deep and three waist-deep). As described in Wanjugi
et al. (2018), water samples were collected via standard methods rec­
ommended in Section 9060 of Standard Methods for the Examination of
Water and Wastewater (APHA, 2005). Samples were collected at
approximately 8:30am on each sampling day (Monday through Friday).
The total number of sampling events was 71 at Grant Park, 67 at
Washington Park, and 67 at Edgewater Beach.
2.3. Cultured bacteria and viral fecal indicator datasets
This study uses previously-published data on the concentrations of
cultured FIB (enterococci and E. coli) and coliphage (F+ and somatic)
from Wanjugi et al. (2018). Briefly, cultured E. coli counts (most prob­
able number, MPN/100ml) were obtained using Colilert Quantitray
(Idexx, Westbrook, ME). Cultured enterococci concentrations (colony
forming units, CFU/100ml) were determined by membrane filtration on
mEI agar (USEPA, 2009). A dead-end hollow fiber ultrafiltration with
single agar layer (D-HFUF-SAL) method was used to enumerate F+ and
somatic coliphage (plaque forming units, PFU/L) as described in
M. Cyterski et al.
McMinn et al. (2017b) and the Supplementary Materials of Wanjugi
et al. (2018).
2.4. qPCR-based bacterial fecal indicator measurements
2.4.1. Water filtration and DNA extraction
Water filtration for qPCR testing was conducted on the same com­
posites used for bacterial and viral fecal indicator culture measurements
as described in EPA Method 1611. Briefly, 100 mL of each composite
water sample was filtered through a 47 mm diameter, 0.40 μm pore size
polycarbonate filter (Millipore, Burlington MA) and stored at − 80 ◦ C.
Next, AE buffer (Qiagen, Germantown, MD) containing 0.2 µg/mL
salmon testes DNA (Sigma-Aldrich), was added to each sample and
subjected to bead milling in an eight-place beater (Biospec Products,
Inc., Bartlesville, OK) at the maximum rate for 1 min. DNA was recov­
ered in the supernatant by centrifugation and the clarified supernatant
was directly used as a template.
2.4.2. qPCR analysis
Analyses for enterococci, E. coli and total Bacteroidales gene se­
quences were performed using previously described assays: Entero1a
(USEPA, 2012a, 2012b, 2015a, 2015b); EC23S857 (Chern et al., 2011);
and GenBac3 (USEPA, 2010c), respectively. All assays were multiplexed
with an internal amplification control (IAC) assay. Target sequences
were amplified in 25 µL reactions containing 5 µL of sample DNA extract,
1 µM of each forward and reverse primer, 80 nM each of the FIB target
sequence and IAC probes, 5 µg of bovine serum albumin (Sigma-Al­
drich), 1X TaqMan Environmental Master Mix (Thermo Fisher Scientific,
Microbiology Division, Lenexa, KS) and ~100 copies per reaction of the
multi-target IAC plasmid DNA control template (USEPA, 2013a, 2015a,
2015b). The salmon testes DNA spiked into extracts was amplified using
the Sketa22 assay (USEPA, 2012a) as described above to evaluate DNA
recovery and monitor for PCR amplification interference. All reactions
were run in a StepOnePlusTM Real-Time PCR System (Applied Bio­
systems, Foster City, CA) with initial denaturation at 95 ◦ C for 10 min
followed by 40 cycles of 95 ◦ C denaturation for 15 s and 60 ◦ C annealing
for 1 min, except for the EC23S857/IAC and corresponding Sketa22
reactions which were annealed at 56 ◦ C. Fluorescence thresholds were
set at 0.03 ΔRN and baseline cycles were determined using the soft­
ware’s AUTO feature.
Blank filter samples (negative controls) containing no target organ­
isms (i.e., negative controls) were prepared in triplicate with each batch
of test sample and analyzed in the same manner as test samples. Whole
cell calibrators (positive control samples) were also prepared in tripli­
cate with each sample batch and identically analyzed. Enterococci and
total Bacteroidales gene copies in the test samples were quantified as
calibrator sequence equivalents (CSEs) using the Δ-Δ comparative Ct
method (USEPA, 2015a, 2015b). The workbook is available (Lane et al.,
2020) with the following modification: median estimates of target se­
quences recovered from the calibrator samples were determined in
advance using a weighted linear regression standard curve model and
the composited Ct measurement data of plasmid DNA standards from
multiple instrument runs as described in Lane et al. (2020). Cultured
Bacteroides thetaiotaomicron (ATCC #29741) and Enterococcus faecalis
(ATCC # 29212) cells were used to prepare the calibrator samples as
described in EPA Method B and EPA Methods 1611, 1609, 1611.1 and
1609.1, respectively. Calibrator samples also contained E. coli (NCTC
#12923) cells but were used only as positive controls for the E. coli
method. E. coli gene copies in the test samples were directly quantified
using the same standard curve model with composited Ct measurement
data generated from analyses of the same multi-target plasmid DNA
standards used for the Entero1a and GenBac3 assays (Sivaganesan et al.,
2018) using a prototype of the automated Excel analysis workbook
presented by Lane et al. (2020). Delta Ct adjustments from the Sketa22
assay were further used in the E. coli workbook to adjust for DNA re­
covery in the test sample extracts as described by Aw et al. (2019).
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Water Research 223 (2022) 118970
Enterococci CSE estimates were converted in the Method 1611.1/1609.1
workbook to calibrator cell equivalents (CCE) for comparisons with
published EPA BAVs (Haugland et al., 2014; USEPA, 2015a, 2015b). All
test sample results used in this study were reported per 100 mL of water
sample, with the log10 copy/reaction quantitative estimates generated
by the E. coli workbook scaled accordingly.
2.4.3. Data acceptance metrics
Each of the Excel data analysis workbooks referenced above per­
formed automatic checks on standard curves, positive and negative
controls, and test sample data quality, and for unacceptable matrix
interference in test samples. In the enterococcus and total Bacteroidales
method workbooks, these checks included: (1) An analysis of covariance
(ANCOVA) with an acceptance criterion of p > 0.05 for slopes and in­
tercepts of the individual standard curves contributing to the composite
curve; (2) target organism and Sketa22 assay Ct measurements for each
of the calibrator or positive control sample analyses performed in each
test sample run within +/− 3 standard deviations of the means deter­
mined for these assays in preliminary analyses; (3) Ct values of duplicate
Sketa22 assay analyses of test samples within 3 units of the mean from
calibrator sample analyses; (4) Ct values of duplicate IAC assay analyses
of test samples within 1.5 units of the mean from negative control
sample analyses; and (5) average target organism CSE estimates for the
negative control (filter blank) samples analyzed in each test sample run
< lower limit of quantification (LLQ) value of 720 prior to Sketa22 assay
delta Ct adjustments. Undetected Ct measurements were assigned values
of 40 for purposes of averaging. The LLQ value established for the EPA
enterococci method is 568 CSE (USEPA, 2013b), however, the 720
CSE/sample value was selected as the LLQ for this study based on the
analyses of plasmid DNA standards with lowest concentration of 6
copies/reaction and 1/120th of total DNA extract volumes analyzed.
Similar data acceptance metrics were applied in the E. coli method
workbook with the following differences: (1) target organism and
Sketa22 assay Ct measurements for each of the positive control sample
analyses performed in each test sample run within acceptance bounds
established from a multiple laboratory evaluation study of the method
(Sivaganesan et al., 2019); (2) mean Ct measurements for the negative
control (filter blank) samples analyzed in each test sample run > 35.09
LLQ Ct estimate established from the composite standard curve in the
workbook (corresponding to 679 copies/sample); (3) standard deviation
of duplicate EC23S857 Ct measurements for test samples that were >
LLQ within 1.414 (Sivaganesan et al., 2019).
2.5. Covariate data
Ancillary water and site characteristics were measured as potential
statistical covariates. These measurements are the same data as pre­
sented in Wanjugi et al. (2018). Instrumentation devices and measure­
ment protocols are described in Table S1 of Wanjugi et al. (2018). All
measured covariates are routinely collected for statistical modeling of
water quality (Francy et al., 2013; USEPA, 2010a). Water parameters
included: water temperature (◦ C), turbidity (NTU), dissolved oxygen
(mg/L), conductivity (μmhos/cm), pH, ultraviolet absorbance in the
water column at 254 nm (UV_254, 1/m), and dissolved organic carbon
(DOC, mg C/L). Site parameters included: wind speed (km/h), wind
direction (ø), air temperature (◦ C), wave height (m), relative humidity
(%), cumulative rainfall (24 h, 48 h, and 72 h; mm), photosynthetically
active radiation (PAR, mol/m2-s), and counts of humans, birds and dogs
at the time of sampling. At Edgewater Beach, the discharge from the
Cuyahoga River was available from a permanent gauging station at its
mouth (USGS site 04208000). For this analysis, wind speed, wind di­
rection and beach orientation angle were converted into alongshore
(Wind-A) and onshore/offshore (Wind-O) wind components using sine
and cosine functions (Cyterski et al. 2013). A correlation analysis
identified multiple highly collinear covariate combinations (r ≥ 0.8):
UV_254 and DOC (r = 0.81), 24 h and 48 h cumulative rainfall (r =
M. Cyterski et al.
0.84), and 48 h and 72 h cumulative rainfall (r = 0.89). To minimize
collinearity for regression coefficient estimation, UV_254 and 48h
rainfall covariates were excluded from further analyses.
2.6. Data analyses
2.6.1. Modeling scenarios and data transformations
Regression models were generated for the seven microbial in­
dicators: F+ coliphage, somatic coliphage, cultured E.coli, cultured
enterococci, qPCR-based E.coli, qPCR-based enterococci, and qPCR-
based Bacteroidales. Prior to any data analyses, cultured FIB and qPCR-
based concentrations (C) were log10 transformed due to a large dy­
namic range with these datasets. Coliphage concentrations were trans­
formed as log10(C+1); the small constant added to prevent negative
log10 values as some coliphage concentrations were <1.0. A value of ½
the detection limit (coliphage) or LLQ (qPCR) was used for microbial
indicator concentrations under the detection limit or LLQ, respectively.
All covariate data were standardized (subtracting the mean and dividing
by the standard deviation).
2.6.2. Microbial indicator measurement correlations
Pearson correlation coefficients (r) were used to examine the
strength of associations between paired microbial indicator measure­
ments by site. To account for potential variability due to occurrence of
non-detects in some data sets, correlation analyses were repeated 100
times for each paired measurement combination and an average r was
calculated. For each of the 100 coefficients, microbial indicators below
the detection limit (coliphage and cultured FIB) or LLQ (qPCR targets)
were assigned a unique uniform random number between zero and the
respective detection limit/LLQ. Significance of the correlation co­
efficients was assessed using a t-test (Kendall and Stuart 1973).
2.6.3. Model formulation
All regression models were developed using the “lars” package
(version 1.2, Efron et al. ,2004) in R (version 4.1.3, R_Core_Team, 2021).
This package implements an iterative fitting technique (LARS-lasso,
Tibshirani, 1997), where the linear regression coefficients are manipu­
lated across successive steps, and Cp (Mallows 1973) is tracked. The
“optimum” model is determined by the step where Cp is minimized. This
implementation is an L1 regularization technique, in which it is possible
for regression coefficients for specific covariates to shrink to zero, in
essence removing them from the model (i.e., there is no evidence that
they are useful).
2.6.4. Model predictive performance evaluation
A cross-validation approach was used to assess predictive perfor­
mance of each site-specific microbial indicator model. For each model,
corresponding data were randomly split into ten folds. Each fold was
withheld while the other nine folds were used to train a sub-model. Each
sub-model was then used to predict microbial indicator measurements in
the withheld data fold. Thus, ten sub-models were developed, resulting
in a single prediction for every data point, as each data point occurs in a
withheld data fold only once. Predicted microbial indicator values from
each set of ten sub-models was used to compute a standardized root
mean squared error of prediction (SRMSEP):
√̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅
∑nm / paired with qPCR-based E. coli, cultured E. coli paired with qPCR-based
2
im=1 (Pim − Oim )
SRMSEPm = Cm (1)
nm
For each model, nm is the number of observations, Pim is the ith
prediction, Oim is the ith observed value, and Cm is the log10 average
concentration of the modeled microbial metric. Along with a SRMSEP,
R2PRED was calculated via a regression using Pim (predicted values) to Oim
(observed values) for each model. For each model, SRMSEP and R2PRED
values were also standardized by dividing a given model performance
metric by the maximum value observed across all models. Overall
4
Water Research 223 (2022) 118970
Performance for each model was then estimated as follows: [standard­
ized R2PRED + (1 – standardized SRMSEP)]. Standardized SRMSEP was
subtracted from one because a high value indicates poor performance.
2.6.5. Covariate evaluation
To assess covariate influence, a single LARS-lasso model was
generated for each site and response variable combination using all
available measurements for each data set. From this model output, only
the regression coefficients were used; models were not used to evaluate
predictive performance. For each site, regression coefficients for each
microbial indicator and covariate combination (7 microbial indicators,
16 covariates) were displayed as heatmaps (“image” function in base R
with color gradient created via the “RColorBrewer” package). The
overall influence for each covariate was then evaluated for each co­
variate across all microbial indicator and site combinations (n = 21)
based on the following metrics: (1) sum of regression coefficients (ab­
solute values used); (2) frequency of non-zero regression coefficients;
and (3) Total Score (sum of metrics 1 and 2). In this analysis, the in­
fluence of 24 h and 72 h cumulative rainfall were summed and called
“Rainfall.” In the same way, the influence of Wind-A and Wind-O were
summed, and this summed influence was called “Wind Speed/
Direction.”
3. Results
3.1. FIB qPCR measurements
Overall percentages of water sample measurements that gave < LLQ
concentration estimates for the Entero1a (enterococci), EC23S857
(E. coli), and GenBac3 (Bacteroidales) assays were 27.2, 8.1 and 6.2,
respectively. Positive and negative control sample acceptance criteria
were met in all 50 instrument runs of the test samples for each method
from the study (data not shown). Each sample was also evaluated for
suitable DNA recovery and absence of amplification interference. These
quality controls indicated that the percentage of test sample analyses
that failed to meet acceptance criteria in the enterococci, E. coli and total
Bacteroidales methods were 2.25, 2.62 and 3.00, respectively. Composite
standard curve performance metrics are summarized in Table 1. Indi­
vidual qPCR measurements for the three study sites are shown in Fig. 2.
3.2. Microbial indicator summary statistics and correlations
Microbial indicator measurements targeting two coliphage (somatic
and F+), two cultured FIB (enterococci, E. coli), and three FIB genetic
markers (enterococci, E. coli, and Bacteroidales) were used as response
variables in LARS models. Table 2 shows summary statistics of each data
set including: number of total samples, number of sample non-detects
and below detection limit (coliphage and cultured FIB) or LLQ (qPCR),
number of samples that failed quality controls, and descriptive statistics
excluding non-detects [minimum, maximum, mean, standard deviation,
and coefficient of variation]. Pearson correlation coefficients (r) be­
tween microbial measures varied by site (Fig. 3). Overall, Edgewater
Beach showed the most significant correlations (11) of the site-specific
datasets, while Washington Park showed the least (5). There were
only a few coefficients that were significant at every site: cultured E. coli
Table 1
Composite qPCR standard curve performance metrics.
Method Standard Curve Amplification Efficiency*
Slope Intercept
Entero1a − 3.49 37.93 0.93
EC23S857 − 3.58 37.78 0.90
GenBac3 − 3.54 37.88 0.92
*
Amplification efficiency = [10(-1/slope)− 1]
M. Cyterski et al. Water Research 223 (2022) 118970

Fig. 2. Raincloud plots showing individual qPCR measurements (log10 concentration) for Edgewater Beach (Panel A), Grant Park (Panel B), and Washington Park
(Panel C) study sites. Open circles show measurements below and shaded circles indicate measurements above the respective detection limit/lower limit of quan­
tification (LLQ). The right/left boundary of each box show the 25th/75th percentiles of the data distribution. The box’s whiskers extend to any observation within 1.5
times the interquartile range. Shaded curves represent respective density distributions.

5
M. Cyterski et al. Water Research 223 (2022) 118970

Table 2
Summary statistics for microbial indicator log10 concentrations measured at the three beach sites. ND = non-detect. Units of measurement for each response and
detection limits/lower limit of quantification (LLQ) are given below the table.
Coliphage Cultured qPCR

Edgewater Beach Fþ Somatic E. coli Enterococci E. coli Enterococci Bacteroidales

Total Measurements 64 64 66 66 67 67 67
Non-Detect/Below LLQ 15 0 0 0 1 15 0
Failed QC** 0 0 0 0 3 3 5
Minimum ND 0.93 1.20 0.60 ND ND 3.27
Maximum 2.03 3.83 3.41 3.61 5.26 4.08 5.89
Mean* 0.42 2.30 2.24 1.85 3.84 2.28 4.75
Standard Deviation* 0.62 0.59 0.50 0.59 0.51 0.68 0.47
Coefficient of Variation 1.50 0.26 0.22 0.32 0.13 0.30 0.10

Grant Park Fþ Somatic E. coli Enterococci E. coli Enterococci Bacteroidales

Total Measurements 69 69 71 71 68 68 68
Non-Detect/Below LLQ 16 1 0 0 5 9 0
Failed QC** 0 0 0 0 2 2 2
Minimum ND ND 1.00 0.30 ND ND 3.99
Maximum 1.38 3.08 3.23 3.79 4.84 3.47 6.25
Mean* 0.26 1.66 1.71 1.55 3.56 2.27 4.87
Standard Deviation* 0.51 0.61 0.65 0.70 0.53 0.53 0.47
Coefficient of Variation 1.94 0.37 0.38 0.45 0.15 0.23 0.10

Washington Park Fþ Somatic E. coli Enterococci E. coli Enterococci Bacteroidales

Total Measurements 62 62 64 64 67 67 67
Non-Detect/Below LLQ 18 3 0 0 6 25 7
Failed QC** 0 0 0 0 1 0 0
Minimum ND ND 0.79 1.00 ND 1.38 2.56
Maximum 1.50 3.06 3.32 4.05 4.73 3.65 5.07
Mean* 0.23 1.63 1.81 1.92 3.34 1.89 3.73
Standard Deviation* 0.49 0.73 0.48 0.59 0.42 0.52 0.60
Coefficient of Variation 2.17 0.45 0.26 0.31 0.13 0.28 0.16
Units PFU/L PFU/L MPN/100mL CFU/100mL Gene Copies/100mL CCE/100mL CSE/100mL
Detection Limit/LLQ − 0.097 − 0.097 0 0 2.83 1.68 2.86
*
non-detections were set to ½ the detection limit (shown at the bottom of the table) for calculations.
**
presumptive sample matrix interference.

enterococci, and qPCR-based E. coli paired with qPCR-based
enterococci.
3.3. Predictive Model Performance
Predictive performance metrics for each model (n = 21) are given in
Table 3. The top performing model (Fig. 4) used the enterococci qPCR
Edgewater Beach data set, yielding an R2PRED of 0.52 and SRMSEP of 0.13
(Overall Performance = 1.87). Seventy one percent of Edgewater Beach
models (5 of 7) occurred in the top 10 based on the Overall Performance
metric. In contrast, 71% (n = 5) of Washington Park models yielded
Overall Performance scores in the bottom seven, including the poorest
performing model (F+, Overall Performance = 0.11). F+ coliphage
models exhibited the poorest performance (Overall Performance ≤
0.31), regardless of site. In addition, all E. coli qPCR models resulted in
Overall Performance values ranked in the bottom 10, while enterococci
qPCR models exhibited the opposite trend (Overall Performance scored
ranked in the top 10) including the top performing model. Somatic
coliphage model Overall Performance scores ranked 3rd (Edgewater
Beach), 7th (Grant Park), and 18th (Washington Park).
3.4. Covariate influence
LARS regression coefficients for each covariate and model combi­
nation are shown in Fig. 5. Model covariate regression coefficients
ranged from − 0.173 (Bacteroidales qPCR, Edgewater Beach) to +0.327
(enterococci qPCR, Washington Park). Discharge from the Cuyahoga
River at Edgewater Beach was the only covariate with regression co­
efficients not equal to zero regardless of microbial indicator model (this
covariate only available for Edgewater Beach). The somatic and F+
coliphage models at Washington Park were the only instances where all
covariate regression coefficients were equal to zero. In contrast, the
enterococci qPCR model for Edgewater Beach was the only occurrence
where all covariates yielded regression coefficients not equal to zero.
The aggregate influence of each covariate across all models is summa­
rized in Table 4. Of the covariates available across all three sites, rainfall
exerted the most influence with non-zero regression coefficient values in
52% (11 of 21) of models. Bird abundance, wave height, and wind
speed/direction were the next most influential covariates across all
microbial indicator models (Total Score ≥ 1.53). Five covariates exerted
minimal influence on predictive models (Total Score ≤ 0.74) with pH
contributing the lowest (Total Score = 0.29).
4. Discussion
4.1. Predicting bacterial and viral fecal indicator concentrations in Great
Lake recreational waters
This study reports the use of LARS-lasso modeling to predict con­
centrations of coliphage (F+ and somatic), cultured FIB (E. coli and
enterococci), and qPCR-based genetic markers (E. coli, enterococci and
Bacteroidales) using water and beach site covariate measurements
collected five-days per week over an entire beach season from three
Great Lake recreational sites. Due to the limited size of recreational
beach season data sets including those reported here, the conventional
practice of parsing a dataset into training and testing subsets was not
feasible. Instead, a cross-validation approach was used to compare
model predictive performance. Findings identified multiple trends. First,
a clear difference in predictive model performance was observed be­
tween F+ and somatic coliphage types. F+ coliphage models consis­
tently resulted in poor predictive performance, ranking the lowest of all
microbial fecal indicators at each recreational beach site. The reduced
performance of F+ coliphage models could be due, in part, to a higher
incidence of non-detects (25.1% for F+ compared to 2.5% for somatic)

6
M. Cyterski et al. Water Research 223 (2022) 118970

Fig. 3. Average Pearson correlation matrices (r) for microbial measures at each study site (A = Edgewater Beach, B = Grant Park, C = Washington Park). Averaged r
values appear in the lower left portion of each panel; t-test derived p-values are given in the corresponding upper right cells. Significant coefficients (p < 0.05) are
bolded. Standard deviations for these coefficients (calculated from 100 simulations where concentrations below detection/LLQ were replaced by random uniform
numbers) never exceeded 0.004 and were primarily < 0.001.

7
M. Cyterski et al. Water Research 223 (2022) 118970

Table 3
Predictive performance metrics for each microbial indicator and site model.
Site Microbial Indicator R2PRED Standardized R2PRED SRMSEP* Standardized SRMSEP Overall Performance

Edgewater Beach F+ Coliphage 0.01 0.02 1.19 0.71 0.31

Grant Park 0.01 0.02 1.50 0.90 0.12
Washington Park 0.06 0.11 1.67 1.00 0.11
Edgewater Beach Somatic Coliphage 0.38 0.74 0.20 0.12 1.61
Grant Park 0.30 0.57 0.30 0.18 1.39
Washington Park 0.06 0.11 0.45 0.27 0.84
Edgewater Beach Cultured E.coli 0.20 0.38 0.14 0.09 1.29
Grant Park 0.31 0.59 0.22 0.13 1.46
Washington Park 0.02 0.03 0.17 0.10 0.93
Edgewater Beach Cultured enterococci 0.40 0.76 0.15 0.09 1.67
Grant Park 0.26 0.50 0.23 0.14 1.37
Washington Park 0.02 0.03 0.20 0.12 0.91
Edgewater Beach E.coli qPCR 0.11 0.21 0.13 0.08 1.13
Grant Park 0.07 0.14 0.14 0.09 1.05
Washington Park 0.01 0.01 0.13 0.08 0.93
Edgewater Beach Enterococci qPCR 0.52 1.00 0.22 0.13 1.87
Grant Park 0.23 0.43 0.21 0.12 1.31
Washington Park 0.18 0.34 0.27 0.16 1.18
Edgewater Beach Bacteroidales qPCR 0.25 0.47 0.09 0.05 1.42
Grant Park 0.09 0.16 0.09 0.06 1.11
Washington Park 0.27 0.53 0.14 0.08 1.44
*
SRMSEP denotes standardized root mean squared error of prediction.

naturalized E. coli populations could obscure any links between

measured covariates and the occurrence of this fecal indicator. Addi­
tional research is warranted to investigate the mechanisms resulting in
poor performance of F+ coliphage and E. coli qPCR fecal indicators.

4.2. Microbial measurement correlation trends

Fig. 4. Scatterplot of qPCR-based enterococci (log10 CCE/100ml) observed
measurements versus LARS sub-model predictions at Edgewater Beach.
and overall lower concentrations in water samples compared to somatic
coliphage. As a result, increasing sample volumes could help alleviate
this issue. However, working with larger volumes (>1 L) presents
additional logistical and expense challenges potentially making this
solution impractical for routine water quality monitoring. In addition,
there is a growing body of evidence suggesting that these virus types
exhibit different occurrence patterns in untreated sewage (Korajkic
et al., 2020), animal fecal samples (McMinn et al., 2014), and across
different surface water types (riverine compared to lake beach) (Wan­
jugi et al., 2018). In contrast, somatic coliphage predictive modeling
exhibited good Overall Performance (Table 3) at Grant Park (1.39) and
Edgewater Beach (1.61), suggesting that this viral indicator could be an
important fecal indicator tool for future water quality forecasting ap­
plications. Second, enterococci and Bacteroidales qPCR outperformed
E. coli qPCR regardless of site. Unlike F+ coliphage, E. coli qPCR
exhibited a reasonable frequency of non-detects across samples (5.9%)
suggesting a different explanation. One possible hypothesis is the pres­
ence of naturalized E. coli populations in soils and beach sand, a phe­
nomenon that has been reported in multiple Great Lake studies (Ishii
et al., 2007; Ishii et al., 2006). The persistence and propagation of
New qPCR findings reported here for E. coli, enterococci, and Bac­
teroidales add to the previously published data set consisting of paired
measurements of cultured FIB and coliphage, providing the opportunity
to evaluate correlation trends between seven different microbial fecal
indicator water quality metrics. Results are particularly useful because
all measurements were generated from the same water sample grabs in
the same laboratory utilizing standardized protocols for each method­
ology. Correlation analyses identified several trends providing potential
insights into the co-occurrence or lack thereof between these water
quality microbial measures. Average correlation coefficients (r) between
cultured E. coli and qPCR measurements ranged from 0.64 (Edgewater
Beach) to 0.87 (Grant Park) and were significant (p < 0.05) regardless of
sampling site. In contrast, a significant correlation between enterococci
measurements was only observed at one site (Edgewater Beach, r = 0.90,
p < 0.001). Previous studies investigating FIB culture and qPCR paired
measurements at Great Lakes beach sites report similar findings, sug­
gesting that the degree of correlation is likely influenced by site specific
conditions (Lavender and Kinzelman 2009, Shrestha and Dorevitch
2019, Whitman et al. 2010). While correlations between culture and
qPCR measurements of E. coli and enterococci are well documented,
little is known regarding the level of agreement between coliphage and
qPCR FIB metrics. In this study, coliphage exhibited markedly lower
correlations with paired qPCR fecal indicator measurements, with most
comparisons resulting in non-significant results (p > 0.05), suggesting
that the occurrence of these viral and bacterial fecal indicators in Great
Lake recreational beach waters are governed by different conditions.
Potential factors might include different animal source shedding pat­
terns or variable fate and transport behaviors. A recent study comparing
coliphage with E. coli qPCR paired measurements in untreated sewage
samples collected across the contiguous United States reported a similar
trend, where somatic coliphage did not significantly correlate with
E. coli qPCR (r = 0.21; p = 0.15), but F+ coliphage exhibited a weak
correlation (r = 0.41, p = 0.003) (Korajkic et al. 2020). Further research
could help elucidate factors contributing to these different occurrence
patterns.

8
M. Cyterski et al. Water Research 223 (2022) 118970

Fig. 5. Heat maps of least-angle regression (LARS) regression coefficients for seven microbial indicator predictive models at Edgewater Beach (Panel A), Grant Park
(Panel B), and Washington Park (Panel C). Covariates are displayed in alphabetical order on the y-axis and microbial measures are shown on the x-axis. Positive and
negative coefficient values are denoted by red and blue shading, respectively. White/pale cells indicate coefficients near or at zero (statistically uninfluential). Cells
shaded in black denote no covariate data available for that site.

(Table 4). Reducing the number of covariate measurements needed to

Table 4
predict water quality would streamline future applications as well as
The influence of each covariate across all models.
lower data collection costs. The analysis of covariate influence also
Covariate Summed Proportion Total provided useful clues about potential fecal sources of pollution at sites.
Coefficients Occurrence Score
For example, bird abundance was the second most influential covariate
Rainfall 1.63 0.52 2.16 across all models (Table 4, Fig. 5), notably at Grant Park and Edgewater
Cuyahoga Discharge* 0.95 1.00 1.95
Beach. Information on potential sources of fecal pollution can help
Bird Abundance 1.23 0.43 1.66
Wave Height 1.13 0.48 1.61
managers identify sites for future microbial source tracking analyses.
Wind Speed/ 1.10 0.43 1.53 Findings also demonstrated the utility of measuring discharge in nearby
Direction lotic hydrologic elements (e.g., the importance of Cuyahoga discharge at
Dissolved Organic 0.87 0.43 1.30 Edgewater Beach). Both Grant Park and Washington Park are also likely
Carbon
influenced by nearby lotic inputs – Oak Creek and Trail Creek, respec­
Turbidity 0.81 0.43 1.24
Relative Humidity 0.75 0.38 1.13 tively – but discharge in these systems was not measured in this study
Dissolved Oxygen 0.51 0.33 0.85 due to the absence of permanent gauge stations. The absence of this
PAR 0.49 0.29 0.77 potentially useful covariate could, in part, have contributed to lower
Water Temperature 0.53 0.24 0.76 model predictive performance at these two sites versus Edgewater
Conductivity 0.50 0.24 0.74
Air Temperature 0.48 0.24 0.72
Beach. Additional research is needed to confirm covariate importance
Dog Abundance 0.38 0.24 0.62 trends identified in this study.
Human Abundance 0.22 0.24 0.46
pH 0.15 0.14 0.29 5. Conclusion
Summed Coefficients are the sum of the absolute values of LARS regression
coefficients in each model. LARS-lasso predictive modeling with cross-validation was used to
Proportion Occurrence denotes the proportion of 21 models where a regression compare the predictive performance of models of coliphage (F+ and
coefficient ∕
= 0. somatic), cultured E. coli and enterococci, and qPCR-based E. coli,
Total Score is the sum of ‘summed coefficients’ and ‘proportion occurrence’. enterococci, and Bacteroidales measures using a suite of environmental
*
Values calculated from seven models, not 21, as this covariate was only
covariates at three recreational beach sites in the Great Lakes basin. Key
measured at Edgewater Beach.
findings include:

4.3. The influence of predictive model covariates
Many covariates were used to predict bacterial and viral fecal indi­
cator concentrations, resulting in several notable trends. Of the cova­
riates used in all models, rainfall was the most influential (Table 4),
echoing findings from other microbial water quality modeling efforts
focused on cultured FIB at recreational beach sites (Nevers and Whit­
man, 2005; Whitman and Nevers, 2008). Findings also lend support to
the development of a potential core set of physical and beach parameter
measurements for future water quality forecasting applications in the
Great Lakes basin, as approximately 25% of the covariate data sets
exerted minimal influence on microbial indicator predictive models
• Models yielded highly variable SRMSEP and R2PRED measures, indi­
cating that some microbial measures may be more amenable to sta­
tistical modeling approaches than others.
• Somatic coliphage models performed at a similar level or better
compared to cultured and qPCR FIB while F+ coliphage models
consistently performed poorly.
• Enterococci and Bacteroidales qPCR outperformed E. coli qPCR
regardless of beach site.
• Rainfall, bird abundance, wave height, and wind speed/direction
were the most influential covariates across all models.
• Approximately 25% of covariates exerted minimal influence on
predictive models suggesting a potential core set of physical and

9
M. Cyterski et al.
beach parameters may be optimal for future water quality fore­
casting applications in the Great Lakes basin.
Additional research is warranted to further characterize the suit­
ability of statistical predictive models for recreational water quality
forecasting of virus and bacterial fecal indicators and confirm trends
observed in this study. Findings also provided useful insights on water
quality forecasting in the Great Lakes demonstrating a challenging re­
ality, there can be a large degree of variability from one site to another.
While LARS allowed for the successful comparison of different microbial
indicator predictive performance and identified important differences
between coliphage types and qPCR-based fecal indicators, future studies
with larger sample sizes could be amendable to alternative approaches
such as machine learning techniques that could further improve water
quality forecasting.
Disclaimer
Information has been subjected to U.S. EPA peer and administrative
review and has been approved for external publication. Any opinions
expressed in this paper are those of the authors and do not necessarily
reflect the official positions and policies of the U.S. EPA. Any mention of
trade names or commercial products does not constitute endorsement or
recommendation for use.
Declaration of Competing Interest
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influence
the work reported in this paper.
Data Availability
Data will be made available on request.
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